The Profile of MicroRNA Expression and Potential Role in the Regulation of Drug-Resistant Genes in Cisplatin- and Paclitaxel-Resistant Ovarian Cancer Cell Lines

Ovarian cancer is the most lethal gynecological malignancy. The high mortality results from late diagnosis and the development of drug resistance. Drug resistance results from changes in the expression of different drug-resistance genes that may be regulated miRNA. The main aim of our study was to detect changes in miRNA expression levels in two cisplatin (CIS) and two paclitaxel (PAC)—resistant variants of the A2780 drug-sensitive ovarian cancer cell line—by miRNA microarray. The next goal was to identify miRNAs responsible for the regulation of drug-resistance genes. We observed changes in the expression of 46 miRNA that may be related to drug resistance. The overexpression of miR-125b-5p, miR-99a-5p, miR-296-3p, and miR-887-3p and downregulation of miR-218-5p, miR-221-3p, and miR-222-3p was observed in both CIS-resistant cell lines. In both PAC-resistant cell lines, we observed the upregulation of miR-221-3p, miR-222-3p, and miR-4485, and decreased expression of miR-551b-3p, miR-551b-5p, and miR-218-5p. Analysis of targets suggest that expression of important drug-resistant genes like protein Tyrosine Phosphatase Receptor Type K (PTPRK), receptor tyrosine kinase—EPHA7, Semaphorin 3A (SEMA3A), or the ATP-binding cassette subfamily B member 1 gene (ABCB1) can be regulated by miRNA.


Introduction
Epithelial ovarian cancer (EOC) is one of the more aggressive gynecological malignancies and is the fifth leading cause of cancer-related deaths in women [1]. According to GLOBOCAN, 295,414 new cases of ovarian cancer and 184,799 of ovarian cancer-related mortality were noted in 2018 [2]. High mortality is related to low diagnosis and development of drug resistance during treatment [3]. Only about 20% of ovarian cancer patients are primarily resistant to chemotherapy, and nearly 80% respond to first-line chemotherapy. However, among these primarily sensitive patients, about 80% will develop drug resistance during treatment [3,4]. This makes the OC an excellent model to study drug resistance development.
The first-line chemotherapy in OC is always composed of platinum compounds (Cisplatin (CIS) or Carboplatin and taxanes (Paclitaxel (PAC)) [5]. CIS is one of the most important drugs used in chemotherapy of many cancers including testis, lung, and ovarian cancer. The mechanism of action of CIS is based on leading to the formation of intrastrand and interstrand DNA cross-linking [6]. It inhibits DNA replication, RNA transcription, and eventually leads to cancer cell death [6,7]. Cancer cells acquire different mechanisms of CIS resistance. The most important is the repair of damaged DNA via DNA repair systems, ovarian cancer to PAC in vitro and in vivo [34]. We also observed the downregulation of miR-31 in two PAC-resistant and CIS-and TOP-resistant cell lines [32]. Downregulation of miR-31 was also observed in CIS-resistant tumors and cell lines of gallbladder cancer (GBC) [35]. miR-30a was significantly downregulated in the PAC-and CIS-resistant EOC cells, and overexpression of miR-30a enhanced sensitivity to CIS in sensitive and resistant cell lines [36]. Downregulation of miR-29a/b/c in CIS-resistant ovarian cancer cell lines has been observed, and the inhibition of miR-29a expression confer CIS-resistance in sensitive cell lines [37]. Previously, we observed strong downregulation of miR-29a in the TOP-resistant cell line and upregulation of its target gene-COL3A1-responsible for TOP-resistance [32]. In PAC-resistant cell line, we observed a correlation between miR-363 downregulation and upregulation of MDR1 gene-encoding glycoprotein P (P-gp) [32].
The miRNA microarray seems to be an effective and accurate tool for miRNA profiling and selecting individual miRNAs involved in the resistance mechanism. In the current study, we used miRNA microarrays to analyze changes in miRNAs expression in CIS-(A2780CR1, A2780CR2) and PAC-resistant (A2780PR1, A2780PR2) ovarian cancer cell lines. We observed both up-and downregulation of the miRNA genes in all drug-resistant cell lines. We also identified many target genes that can be responsible for drug resistance in investigated cell lines.

Gene Chip Quality Assessment
In the present study, we used standard factors such as signal-to-noise ratio internal hybridization and controlled spike-in-controls to determine the quality of analyzed samples preliminarily. Controlled spike-in-controls were spike_in-control-2, spike_in-control-23, spike_in-control-29, spike_in-control-31, spike_in-control-36. Oligos 2, 23, and 29 are RNA, confirming the poly(A) tailing and the ligation. Oligo 31 (poly(A) RNA) confirmed ligation. Oligo 36 is poly(day) DNA and confirmed ligation and the lack of RNases in the RNA sample.

Gene Expression Evaluation and Gene Expression Lists
The current study determined variations in miRNA transcriptional profiles in two CIS-resistant and two PAC-resistant ovarian cancer cell lines. Obtained results provided new information regarding the significance of miRNA expression changes in the resistance development of these drugs in ovarian cancer. miRNA expression changes in CIS-and PAC-resistant sublines in relation to the drug-sensitive A2780 cell line are summarized in Supplementary Table S1. To determine the differentially expressed miRNA, we applied the following cut-off criteria: expression level changes higher than 5-fold or lower than 0.2-fold (up-/downregulation of more than/less than 5 and −5, respectively) and adjusted p-value < 0.05. Comparisons of miRNA expression were performed for the drug-resistant cells versus their drug-sensitive counterparts.

miRNAs Expression in CIS-and PAC-Resistant Cell Lines
The overall miRNA expression profile of the appropriate experimental groups is shown in Figure 1. We observed changes in the expression of 150 miRNAs (Supplementary Table S1). The expression of 129 miRNAs was elevated in at least one drug-resistant cell line. Expression of 18 miRNAs was downregulated in at least one drug-resistant cell line. The expression of three miRNAs was up-or downregulated, depending on the cell line. To make our analysis more specific for resistance to CIS or PAC for further analysis, we selected only miRNAs that were 1. changed at least five-fold (fold ±5, p < 0.05) in two cell lines resistant to the same drug-CIS or PAC, respectively; 2. at least 10-fold changes in miRNA expression in one drug-resistant cell line. As a result, we observed changes in expression of 46 miRNAs (Table 1, Figure 2). The level of expression of 34 miRNAs was upregulated in at least one drug-resistant cell line. Expression of nine miRNAs was Figure 1. Volcano plots displaying the miRNA with expression levels that were upregulated or downregulated (green dots) by 5-fold or more in drug-resistant cells in relation to the A2780 cells.

Figure 1.
Volcano plots displaying the miRNA with expression levels that were upregulated or downregulated (green dots) by 5-fold or more in drug-resistant cells in relation to the A2780 cells.
The expression of three miRNAs was changed in all investigated cell lines, and among them, expression of miR-218-5p was downregulated in all investigated cell lines. Expression of miR-221-3p and miR-222-3p was downregulated in both CIS-resistant cell lines but upregulated in both PAC-resistant cell lines. Expression of five miRNAs was changed in three of four cell lines, and among them expression of miR-125b-5p, miR-887-3p was upregulated in both CIS-resistant cell lines and in the A2780PR2 cell line, expression of miR-296-3p was upregulated in both CIS-resistant cell lines and in the A2780PR1 cell line, expression of miR-99a-5p was upregulated in both CIS-resistant cell lines but downregulated in the A2780PR2 cell line, and expression of miR-551b-3p was downregulated in both PAC-resistant cell lines and the A2780CR1 cell line. Additionally, miR-551b-5p was downregulated in both PAC-resistant cell lines and expression of miR-4485 was upregulated in both PAC-resistant cell lines Thus, the upregulation of miR-125b-5p, miR-99a-5p, miR-296-3p, miR-887-3p, and miR-4485, and downregulation of miR-218-5p, miR-221-3p, and miR-222-3p, was specific to CIS-resistance. On the other hand, upregulation of miR-221-3p and miR-222-3p, and downregulation of miR-551b-3p, miR-551b-5p, and miR-218-5p, was specific for PAC-resistance.    Expression of another 37 miRNAs was changed at least 10-fold in one resistant cell line. Among them, expression of 31 miRNAs was upregulated, and expression of 6 miRNAs was downregulated.

Analysis of Target Genes Expression
We further examined whether differentially expressed miRNAs are involved in the regulation of genes responsible for the drug resistance development. Based on the assumption that an increase in miRNA expression causes a decrease of target gene expression and vice versa, for further analysis, we selected only those target genes for which the fold change value was inversely correlated with miRNA fold change. We applied the same cut-off values for both miRNAs and their targets-at least 5-fold up/down and adjusted p value <0.05. Target expression below cut-off criteria was considered as "not significant (NS)" when the target gene lists were constructed. We used our microarray data, which has been published previously [24][25][26]. Using these criteria (changes in expression 5-fold up or down), we found targets for 106 from 150 miRNAs.
For further analysis, target genes related to drug resistance, extracellular matrix, and cancer stem cell biology were selected from the whole population of target genes using the following key words in the Gene Ontology (GO) database: response to a drug, drug transport, extracellular space, extracellular matrix, collagen-containing extracellular matrix, a stem cell. Previously, we described that these genes belong to differentially expressed genes in drug-resistant cell lines [24][25][26].
The second drug used in the first line of ovarian cancer chemotherapy is PAC. Thus, we also analyzed miRs expression in two PAC-resistant cell lines. In the A2780PR1 cell line, we identified targets for 39 miRNAs ( Figure 5). LAMB3 overexpression correlated with downregulation of miR-218-5p. In contrast to CIS-resistant cell lines, ROBO2 overexpression correlated with downregulation of miR-218-5p. We also observed that TNFSF10 gene downregulation correlated with overexpression of miR-221-3p and miR-222-3p. We also observed regulation of SLC transporters by miRNAs. Among them, miR-218-5p downregulation correlated with SLC16A14 upregulation, and miR-8064 upregulation correlated with SLC27A2 downregulation. As increased protein phosphorylation is a key feature of drug-resistant cells, we also observed downregulation of protein phosphatases by miRNAs. PTPRK was regulated by miR-1910-5p and miR-767-5p, and PTPRZ1 was regulated by miR-129-5p, miR-6800-5p, and miR-200c-3p. In contrast, upregulation of EPHA7 gene-encoding protein kinase (EPH Homology Kinase 3) correlated with downregulation of miR-196a-5p, miR-196b-5p, and miR-218-5p. We also observed increased expression of genes involved in cancer stem cells biology. Upregulation of ALDH1A1 (Aldehyde Dehydrogenase 1 Family Member A1) resulted from miR-551b-5p downregulation. LOX (Lysyl Oxidase) upregulation correlated with downregulation of miR-218-5p and miR-3613-3p. Additionally, downregulation of miR-3613-3p correlated with expression of other drug-resistant genes: SAMD4A (Sterile Alpha Motif Domain Containing 4A) and RUNDC3B (RUN Domain-Containing Protein 3B). Additionally, expression of SAMD4A was also regulated by miR-551b, and miR-10a-5p and miR-10b-5p regulated expression of RUNDC3B. Decreased expression of SEMA3A (Semaphorin 3A) resulted from increased expression of miR-145-5p. miR-222-3p. Decreased expression of the ROBO2 gene inversely co upregulation of miR-145-5p. Similar to previously, we also observed dow SLC transporters. SLC1A1 downregulation correlated with miR-145-5p decreased expression of SLC43A1 correlated with upregulation of upregulation of let-7c-5p correlated with downregulation of SLC6A15, and of miR-628-3p correlated with downregulation of SLC40A1. In this cell observed downregulation of tyrosine phosphatases. Among them, PTPRK by miR-409-3p, PTPRD (Protein Tyrosine Phosphatase Receptor Type D) by PTPRZ1 (Protein Tyrosine Phosphatase Receptor Type Z1) by miR-431-5p. The second drug used in the first line of ovarian cancer chemotherapy we also analyzed miRs expression in two PAC-resistant cell lines. In the line, we identified targets for 39 miRNAs ( Figure 5). LAMB3 overexpress with downregulation of miR-218-5p. In contrast to CIS-resistant cell overexpression correlated with downregulation of miR-218-5p. We also TNFSF10 gene downregulation correlated with overexpression of miR-22 222-3p. We also observed regulation of SLC transporters by miRNAs. Amo 218-5p downregulation correlated with SLC16A14 upregulation, a upregulation correlated with SLC27A2 downregulation. As incre phosphorylation is a key feature of drug-resistant cells, we also observed do of protein phosphatases by miRNAs. PTPRK was regulated by miR-1910-5p 5p, and PTPRZ1 was regulated by miR-129-5p, miR-6800-5p, and m contrast, upregulation of EPHA7 gene-encoding protein kinase (EPH Hom correlated with downregulation of miR-196a-5p, miR-196b-5p, and miR-21 observed increased expression of genes involved in cancer stem Upregulation of ALDH1A1 (Aldehyde Dehydrogenase 1 Family Member A1) miR-551b-5p downregulation. LOX (Lysyl Oxidase) upregulation co downregulation of miR-218-5p and miR-3613-3p. Additionally, downregu 3613-3p correlated with expression of other drug-resistant genes: SAMD4A Motif Domain Containing 4A) and RUNDC3B (RUN Domain-Containin Additionally, expression of SAMD4A was also regulated by miR-551b, an In the second PAC-resistant cell line, we identified targets for 13 miRNAs (F Increased expression of COL4A1 (Collagen Type IV Alpha 1 Chain) resulte downregulation of miR-99a-5p. Similar to in the A2780PR1 cell line, we also obser TNFSF10 downregulation correlated with overexpression of miR-221-3p and miR As in other resistant cell lines, we also observed decreased expression of phosphatases that correlated with increased expression of specific miRNAs. In PTPRK was regulated by miR-149-5p, and PTPRD by miR-339-5p. In contrast, kinase EPHA7 was increased and correlated with miR-18b-5p and miR downregulation. We also observed increased expression of SAMD4 associat decreased expression of miR-19a-3p and miR-551b-5p, and increased expres RUNDC3B associated with miR-99a-5p. The most important protein responsible f resistance in this cell line is glycoprotein P (P-gp) encoded by the MDR1 (Multidrug Resistance Protein 1/ ATP Binding Cassette Subfamily B Member 1) gene. W observe that a strong increase in ABCB1 expression correlated with decreased exp of miR-21-5p.  In the second PAC-resistant cell line, we identified targets for 13 miRNAs (Figure 6). Increased expression of COL4A1 (Collagen Type IV Alpha 1 Chain) resulted from downregulation of miR-99a-5p. Similar to in the A2780PR1 cell line, we also observed that TNFSF10 downregulation correlated with overexpression of miR-221-3p and miR-222-3p. As in other resistant cell lines, we also observed decreased expression of protein phosphatases that correlated with increased expression of specific miRNAs. In detail: PTPRK was regulated by miR-149-5p, and PTPRD by miR-339-5p. In contrast, protein kinase EPHA7 was increased and correlated with miR-18b-5p and miR-218-5p downregulation. We also observed increased expression of SAMD4 associated with decreased expression of miR-19a-3p and miR-551b-5p, and increased expression of RUNDC3B associated with miR-99a-5p. The most important protein responsible for PAC-resistance in this cell line is glycoprotein P (P-gp) encoded by the MDR1/ABCB1 (Multidrug Resistance Protein 1/ ATP Binding Cassette Subfamily B Member 1) gene. We could observe that a strong increase in ABCB1 expression correlated with decreased expression of miR-21-5p.

Discussion
The development of drug resistance is a significant problem in ovarian cancer chemotherapy. Hundreds of genes are known to play a role in this process, and new ones are still being described. Among mammalian genes, up to 60% can be regulated by miRNA [22]. As miRNAs are considered a new therapeutic tool in many diseases, including cancer [38], the knowledge about the regulation of drug-resistance genes by miRNAs seems to be very important from a clinical point of view.
The present results show the correlation between resistance to cytotoxic drugs and the expression of genes encoding microRNAs. Here, we analyzed the expression of miRNA genes in ovarian cancer cell lines resistant to CIS and PAC-drugs used in first-line chemotherapy of OC [5]. In contrast to studies where only one pair of sensitive/resistant cell lines is tested, in this study, changes in miRNA expression in four drug-resistant cell lines derived from one parental cell line were examined. Our results also compared miRNA expression in twin cell lines resistant to CIS and PAC. It is a unique model for such research.
In these cell lines, we previously observed increased expression of genes known from resistance to CIS, such as MRP2 in both CIS-resistant cell lines, or PAC, such as MDR1/P-gp in both PAC-resistant cell lines [39,40]. We also observed changes in the expression of many genes encoding ECM molecules [41]. Additionally, we noted up-or downregulation of "new drug-resistant genes" that may be related to CIS and/or PAC resistance, as the changes were observed in three cell lines resistant to the same drug [42][43][44]. Additionally, in drug-resistant cell lines, we observed population of ALDH1A1-positive cancer stem cells (CSCs) [45,46]. However, we did not observe any cross-resistance to PAC in CIS-resistant cell lines, and there was a low level of resistance to CIS in the A2780PR2 cell line [39]. To make our analysis more specific, we started our investigation from miRNA changes in two cell lines resistant to CIS or PAC, respectively. Among 46 miRNAs analyzed in this experiment, the expression of four (miR-125b-5p, miR-99a5p, miR-296-3p, and miR-887-3p) was upregulated and three (miR-218-5p, miR-221-3p, and miR-222-3p) was downregulated in both CIS-resistant cell lines, suggesting their role in CIS resistance. In contrast to our study, the research conducted on colon cancer [47] and gallbladder cancer [48] pointed to downregulation of miR-125b-5p in CIS-resistant models and suggested its suppressor role in resistance to CIS both in vitro and in vivo. The differences between these and our results can result from a different mechanism of CIS resistance in investigated cell lines. Both CIS-resistant cell lines were also characterized by overexpression of miR-99a-5p. The results in line with our research were presented on gastric cancer (GC). Significant upregulation of miR-99a-5p was observed in CIS-resistant GC tissues as well as in CIS-resistant cell lines [49]. An elevated level of miR-99a-5p was also observed in serum from ovarian cancer patients and promoted cancer cells invasion in vitro [50]. Thus, miR-99a-5p upregulation seems to be related to OC invasion and drug resistance. The downregulation of miR-218-5p resulted in increased resistance to CIS in this cancer, as well [51]. In ovarian cancer study, decreased expression of miR-218-5p was observed in ovarian cancer tissue in comparison to normal ovary and in ovarian cancer cell lines in comparison to normal ovarian cell lines. Furthermore, decreased level was observed in patients with FIGO III, IV compared to with FIGO I, II and in patients with the presence of metastasis, suggesting a significant role of miR-218-5p downregulation in ovarian cancer progression. As metastasis are usually more resistant than primary tumors, and we observe reduction of miR-218-5p expression in drugresistant cell lines, we can suggest that this miR can be associated with both processes [52]. The last two miRs downregulated in both CIS-resistance cell lines were miR-221-3p and miR-222-3p. The relationship between the expression of these miRs and CIS-resistance is not known so far. However, ovarian cancer study has shown that decreased level of both miR-221-3p and miR-222-3p was linked to lower overall survival (OS) of patients [53,54]. Furthermore, more malignant cells showed reduced expression of miR-221-3p [54], which was associated with higher tumor grade (lower in G3 vs. G1, G2) and correlated with tumor growth in vivo [54]. In vitro experiments indicated that miR-222-3p inhibited EOC cell proliferation and migration [54]. In summary, the downregulation of both miRs seems to be related to ovarian cancer progression and CIS-resistance development. Based on the results obtained, we can assume that miR-99a-5p, miR-218-5p, miR-221-3p, and miR-222-3p may play a role in ovarian cancer progression and drug resistance. However, the detail of the role requires functional study.
In contrast to CIS-resistant cell lines in both PAC-resistant cell lines, both miR-221-3p and miR-222-3p were upregulated. We did not find any data concerning miR-222-3p expression in PAC-resistance cancer or cancer cell lines. On the contrary to our results, strong downregulation of miR-221-3p was observed in non-small-cell lung cancer (NSCLC) PAC-resistant cell line (A549/Taxol) compared to parental control [55]. Those differences may be due to other PAC-resistance mechanisms in investigated cell lines. Both strands of miR-551b (miR-551b-3p and miR-551b-5p) had a downregulated expression in both PACresistant cell lines. miR-551b-3p, expression level was also significantly downregulated in the gastric cancer tissues, which correlated with the degree of differentiation (i.e., tumor grade), TNM stage, and lymph node metastasis [56]. Downregulation of miR-551b-5p was reported in breast cancer cell lines and tissues and was an indicator of poor overall survival [57]. We observed downregulation of both miRs in PAC-resistant cell lines while in others during tumor progression. As a tumor progression is usually related to higher drug resistance, miR-551b-3p and miR-551b-5p downregulation can be related to both processes.
Next, we analyzed other miRNAs whose expression was significantly altered in tested drug-resistant cell lines. However, the pattern of their expression differed between cell lines resistant to the same drug. Most highly overexpressed or downregulated miRNA were observed in PAC-resistant cell lines. The highest expression was observed for miR-205-5p in the A2780PR1 cell line (over 500-fold), suggesting its significant role in resistance to PAC. The role of miR-205-5p overexpression in PAC resistance seems to be supported by other studies. miR-205-5p was upregulated in endometrial cancer (EC) tissues and cell lines compared with respective control [58]. Here, we observe increased expression in the A2780 cell line that is isolated from endometrioid ovarian cancer [59]. Hence, the similar pattern of expression can result from similar origin of both cancers. Expression of miR-205-5p was also related to ovarian cancer progression, as plasma level of miR-205-5p was significantly elevated in ovarian cancer patients compared to control and in poorly differentiated tumors compared to well/moderate differentiated ones [60]. Overexpression of miR-205-5p was also elevated in HGSC tumors in comparison to ovarian surface epithelium [61]. Thus, overexpression of miR-205-5p seems to be related to both drug resistance and ovarian cancer progression. The upregulation of miR-200c-3p in PAC-resistant cell lines seems to be following its role in OC progression. The miR-200c-3p level was upregulated in ovarian cancer tissue and serum exosomes and is related to the presence of distinct metastasis [62]. In HGSC patients, high miR-200c-3p expression was associated with poor progressionfree and overall survival [61]. In another HGSC study, increased expression of miR-200c-3p in tumor tissue was observed in stage III/IV, compared to stage I/II [63]. Thus, increased expression of miR-200c-3p may be related to ovarian cancer progression and PAC resistance. An elevated level of miR-100-5p in the A2780PR2 cell line seems to be related to PAC resistance. This miR was also observed in PAC-resistant prostate cancer cell lines [64] and ovarian cancer tissue [65]. Downregulation of miR-10a-5p in the A2780PR1 cell line may be related to PAC resistance in ovarian cancer, as we previously also observed downregulation of miR-10a-5p expression in two other PAC-resistant ovarian cancer cell lines [32]. Expression of miR-10a-5p was downregulated in ovarian cancer cell lines, tissue, and serum, and correlated with FIGO stage (lower in III/IV), presence of lymph node metastasis, and poor OS [66]. Therefore, we hypothesize that it may influence ovarian cancer progression and drug resistance. The last downregulated miR analyzed in this study was miR-383-5p. The upregulation of this miR enhanced the sensitivity of OC cell lines to PAC [67]. Thus, this miR's downregulation should enhance PAC resistance, which we could observe in our study. Summarizing this part of the analysis, we can conclude that changes in the expression of miR-205-5p, miR-200c-3p, miR-100-5p, miR-10a-5p, and miR-383-5p are supported by other results, and we suggest their role in PAC resistance and/or ovarian cancer progression.
Presented results may indicate the potential role of miRNA in drug-resistance development. It concerns especially miRNAs down/upregulated in two cell lines resistant to CIS or PAC. We also observed differences in the expression of particular miRNAs between two cell lines resistant to the same drug. It emphasizes the complexity of the drug-resistance mechanism development, which we could also observe for the protein-encoding genes involved in this process [40][41][42].
Interpretation of miRNA results is much more complicated than of protein-coding genes expression. For example, expression of ABC transporters such as P-gp or BCRP is always related to PAC and DOX or TOP resistance, respectively [12]. miRNA influence is more comprehensive, as one miRNA may regulate the expression of many genes, yet one miRNA can be a target of different miRs. Another issue is that miRNAs do not directly regulate drug-resistance genes expression but modulate the signaling pathways regulating their expression.
To better understand the role of investigated miRNA in drug resistance, the second part of our investigation was devoted to reverse correlation between the expression of miRs and their target genes. We focused mainly on genes previously described by ours in the context of drug resistance [40][41][42].
Previously, both CIS-resistant cell lines presented the upregulation of TIMP3 belonging to tissue inhibitors of metalloproteinases gene family, involved in the degradation of the ECM [41]. Here, the upregulation of TIMP3 correlated with a downregulation of miR-221-3p and miR-222-3p in both CIS-resistant cell lines. Regulation of TIMP3 by miR-221-3p was described in diabetic retinopathy [68] and by miR-221-3p and miR-222-3p chronic liver disease [69]. Overexpression of miR-222-3p was associated with the downregulation of TIMP3 in osteosarcoma and promoted cells proliferation and invasion [70]. Thus, the involvement of miR-221-3p and miR-222-2p seems to be a universal mechanism of TIMP3 gene regulation.
Among other ECM genes regulated by miRNA, we can distinguish CDH2 in the A2780CR2 cell line, LAMB3 in the A2780PR1 cell line, and COL4A1 in the A2780PR2 cell line. CDH2 upregulation was associated with decreased expression of miR-218-5p. Association between CDH2 and miR-218-5p has been reported by others [71]. We could observe increased expression of LAMB3 in PAC-resistant cell lines. Overexpression of LAMB3 was also observed in platinum-resistant ovarian cancer patients [72], suggesting this gene's role in drug resistance. In our study, increased expression of LAMB3 correlated with decreased expression of miR-218-5p. This is supported by head and neck squamous cell carcinoma (HNSCC) study, where direct regulation of LAMB3 by miR-218 has been described and targeting of LAMB3 by miR-218 resulted in significant inhibition of cell migration and invasion [73]. Upregulation of COL4A1 in the A2780PR2 cell line correlated with miR-21-5p downregulation. Regulation of COL4A1 expression by miR-21-5p was described previously in papillary thyroid cancer [74]. ROBO2 is a transmembrane receptor for the Slit homolog 2 protein and functions in axon guidance and cell migration [75]. It is a putative tumorsuppressor gene, and its expression was reduced in different cancers, including ovarian cancer. Decreased expression of ROBO2 was observed in primary cultures of ovarian cancer epithelial cells, compared to normal OSE, and in poorly differentiated SKOV-3 cells, compared to the more differentiated PEO-14 cells [76]. We observed downregulation of ROBO2 in both CIS-resistant cell lines, suggesting its role in resistance to this drug. Regulation of ROBO2 by miR-145-5p was also observed in primary neurons [77]. In contrast, ROBO2 expression was upregulated in the A2780PR1 cell line and was associated with miR-218-5p downregulation. Regulation of ROBO2 by this miR was also noted in vascularization of the retina, where ROBO2 was a direct target of miR-218-5p [78]. We could follow a different pattern of ROBO2 expression in CIS-and PAC-resistant cell lines regulated by miRNA. Kinases and phosphatases activate/deactivate signaling proteins by reversible phosphorylation resulting in up/downregulation signal transduction and changes in genes expression, cellular metabolism, and rate of cell proliferation [79]. The characteristic feature of cancer cells is a high level of protein phosphorylation [80], which plays an important role in the development of drug resistance [81,82]. Changes in protein phosphorylation can be related to increased expression of protein kinases and decreased expression of protein phosphatases. Previously, we observed reduced expression of PTPRK in 17 drug-resistant ovarian cancer cell lines [83]. Here, we observed that in both CIS-and PAC-resistant cell lines, the downregulation of PTPRK correlated with upregulation of different miRNAs. In the A2780CR1 and A2780PR1 cell lines, we observed a relationship between PTPRK downregulation and upregulation of miR-1910-5p. Additionally, in the A2780PR1 cell line, we observed regulation by miR-767-5p. However, in the A2780CR2 cell line, PTPRK was regulated by miR-409-3p, and in the A2780PR2 cell line by miR-149-5p. Thus, expression of the same gene, even in twin cell lines resistant to the same drug, was regulated by different miRNA. Increased signal transduction can also be associated with increased kinases activity and expression. Previously, we described increased expression of EPHA7 (EPH receptor A7) in three PAC-resistant cell lines [42]. EPHA7 encodes the ephrin receptor, which belongs to the protein-tyrosine kinase family and functions as receptor-tyrosine kinase, resulting in activation of ERK pathway [84]. Its increased expression was observed, among others, in gallbladder adenocarcinoma, where it was an independent poor prognostic marker, and its upregulation was associated with carcinogenesis, disease progression, and a poor prognosis [85]. Previously, in another PAC-resistant cell line, we observed that EPHA7 overexpression correlates with downregulation of miR-18b [32], which was also supported by other studies [86]. Here, we noticed that EPHA7 overexpression correlates with miR-18b-5p downregulation in the A2780PR2 cell line. It suggests the role of this miR in EPHA7 gene regulation. Additionally, in the A2780PR1 cell line we observed regulation of EPHA7 expression by miR-196a-5p and miR-196b-5p. Regulation of EPHA7 by miR-196b was also observed in esophageal squamous cell carcinoma [87]. Semaphorins are proteins involved in many developmental processes and cancer progression [88]. The downregulation of SEMA3A as a tumor suppressor gene was described in many cancers, including ovarian cancer [89]. Previously, we described downregulation of SEMA3A expression in three from four ovarian cancer PAC-resistant cell lines [44], and in the W1-PAC-resistant cell line, its expression was regulated by miR-145 [32]. Here, the downregulation of SEMA3A in the A2780PR1 cell line also correlated with miR-145 upregulation. This regulation is also supported by others [90].The most important gene involved in PAC resistance is glycoprotein P, encoded by MDR1/ABCB1 [12]. Its expression was reported by ours previously in all PAC-resistant cell lines [42]. Here, the high upregulation of the MDR1 gene correlated with the downregulation of miR-21-5p. MDR1 has also been identified as a target of miR-21 in colorectal adenocarcinoma [91]. In summary, we have identified many miRNA-target pairs, and some of them were described by others. Other ones, we found for the first time. Furthermore, even the tween twin cell lines resistant to the same drug present different levels of regulation, where one target may be regulated by different miRNA. It proves that the mechanism of action relying on miRNA regulation is complex and complicated.

Cell Lines and Cell Culture
The human ovarian carcinoma A2780 cell line was purchased from ATCC. A2780 sublines that were resistant to CIS (A2780CR1, A2780CR2 (A2780 cisplatin-resistant)), and PAC (A2780PR1 and A2780PR2 (A2780 paclitaxel-resistant)) were obtained by exposing A2780 cells to the relevant drugs at gradually increasing concentrations. The final concentrations used to select the resistant cells were 1000 ng/mL of CIS for both A2780CR1 and A2780CR2 cell lines, 300 ng/mL of PAC for A2780PR1 cell line, and 1100 ng/mL of PAC for A2780PR2 cell line, as described previously [39]. They were twofold greater than the concentrations in the plasma 2 h after intravenous administration [92]. According to parental drug-sensitive cell lines, the increase in resistance was as follows: 4.09-fold for A2780CR1 vs. A2780, 3.29-fold for A2780CR2 vs. A2780, 146-fold for A2780PR1 vs. A2780, and 1202-fold for A2780PR2 vs. A2780, as we described previously [39]. All cell lines were maintained as a monolayer in the complete medium (MEM medium supplemented with 10% (v/v) fetal bovine serum, 2 pM L-glutamine, penicillin (100 U/mL), streptomycin (100 U/mL) and amphotericin B (25 µg/mL)) at 37 • C in a 5% CO 2 atmosphere.

miRNA Isolation
miRNA was isolated using a Qiagen reagent kit following the producer's protocol. The concentration and quality of isolated miRNA was quantified by measuring absorbance at 260 nm and 280 nm. The 260/280 nm ratio was used to estimate the degree of protein contamination. In all samples, the 260/280 nm ratio ranged from 1.8 to 2.0. From each study group, four samples were subjected to miRNA expression profiling by microarray. (N/group = 4).

Microarray Preparation, Hybridization, and Scanning
The profiling of miRNA expression was performed using Affymetrix platform-based microarrays with GeneChip™ miRNA 4.1 Array Strip (Thermo Fisher Scientific, Waltham, MA, USA). The detailed technical procedure is described elsewhere [93,94]. Each microarray was designed following the miRBase Release 20 database, including complementary probes for 2578 human mature miRNA, 1996 human snoRNA, CDBox RNA, H/ACA Box RNA, scaRNA, and 2025 human pre-miRNA. Preparation of miRNA to hybridization step was performed using the FlashTagTM Biotin HSR RNA Labeling Kit (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, 150 ng of miRNA was subjected to the poly(A) tailing and biotin ligation under the producer's protocol. Biotin-labeled miRNA were then hybridized to GeneChip™ miRNA 4.1 Array Strip (20 h, 48 • C). Microarrays were then washed and stained according to the technical protocol using the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). The array strips were scanned using an Imaging Station of GeneAtlas System (Thermo Fisher Scientific, Waltham, MA, USA).

Microarray Analysis and miRNA Gene Screening
The preliminary analysis of scanned microarrays was carried out using Affymetrix GeneAtlas Operating Software (Affymetrix, Santa Clara, CA, USA). The quality of the miRNA expression profile was verified using default quality control criteria determined by the software. BioConductor libraries from the statistical programming language "R" were used for further analyses. The raw CEL files obtained as a result of microarray scanning were imported into this programming environment. The Robust Multiarray Average (RMA) normalization algorithm (implemented in the Affy library) was used for normalization, background correction, and expression calculation [95]. The biological annotation for human miRNA was retrieved from the pd.mirna 4.1 library, where tabulated biological descriptors were merged with a miRNA normalized dataset to obtain a complete miRNA data table. Differential expression and statistical evaluation were determined using the "limma" library based on the linear model for microarray data [96]. p-values were calculated using an empirical Bayes moderated t-test with false discovery rate (FDR) correction for multiple tests. The selection criteria for significantly regulated miRNA expression were based on the difference between folds greater than absolute five and p-value after FDR correction (adj.p.val) < 0.05. Obtained results were visualized as scatter plots showing the total number of miRNAs up-and downregulated. Differentially expressed miRNA were also presented as heat maps and tables. Raw data files were deposited in the Gene Expression Omnibus (GEO) repository at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo/, accessed on 2 December 2021) under the GEO accession number GEO: GSE190245. Public on 2 December 2021.

miRNA-Target Gene Prediction
To identify potential target genes for differently expressed miRNA, we applied the SpidermiR library, where symbols of differentially expressed miRNAs were used to find target genes in the following databases: for predicted targets-DIANA, Miranda, PicTar, TargetScan, and for experimentally validated targets-miRTAR, miRwalk [97]. The mRNA transcriptomic data from our published experiment were used to determine the actual expression value of the target genes [40][41][42]. Obtained fold change values for mRNA were merged with the target genes data table. We selected for further analysis only target genes whose fold change was inversely correlated with the fold change value of appropriate miRNA (cut-off criteria: fold ±5, adjusted p value (adj.p.val.) <0.05). From the entire collection of miRNA-targets pairs, we selected only those that were involved in drug resistance, extracellular matrix, and cancer stem cell biology applying the following GO terms keywords: "collagen-containing extracellular matrix", "extracellular matrix", "extracellular space", "response to drug", and "stem cell". Interactions between miRNA and target genes in relation to selected GO terms were visualized using Cytoscape 3.7.2 (National Institute of General Medical Sciences, Bethesda, MD, USA) [98].

Conclusions
In conclusion, we identified miRNAs that may be specific to CIS or PAC resistance, as their expression changes were observed in both CIS-and PAC-resistant cell lines. Some of them were previously described in the drug resistance or ovarian cancer progression, while the expression of others was observed for the first time. We also noted an inverse correlation between the expression of miRNAs and their targets, for a few pairs observed for the first time. The relation between new pairs needs to be confirmed at a molecular level. The significance of this regulation must be explained in the context of the resistance to cytotoxic drugs.